Grow bacteria to OD600 0.5
Spin at 4000rpm (3250g) for 7 min at room temp.
Resuspend in wash buffer
10mM Tris pH 7.4
2.5mM MgCl2
1X PBS
10% glycerol
500ml wash buffer
100ml 10mM Tris pH 7.4
12.5 ml of 100 mM MgCl2
50 ml 10X PBS
50 ml 100% glycerol
Washand spin at 9000rpm and 4C for 5min
Wash and spin again at 9000rpm and 4C for 5 min
Resuspend in 4ml (or 30 ml) lysis buffer and freeze at -80C.
This process needs to be as quick as possible.
Lysis buffer:
10mM Tris pH 7.4
5mM KCl
1.5mM DTT
Complete PI cocktail (Roche) 1X; EDTA free.
DNaseI 1X
RNase 1X
bacterial samples from the -80 and allowed to thaw on ice.
Samples subjected to the French Press 3-4X each.
Add EDTA to 50mM
Low speed spin 8000 rpm for 10 min @ 4C to remove the soluble fraction.
High speed spin at 50000g for 1 hour in the ultracentrifuge to remove the membrane fraction.
Samples frozen at -80
Bradford protein assay
Standard curve was generated as follows:
1, 2, 4, 8, 16 ul of 2mg/ml BSA (biorad) were added to sterile MQ water to achieve 20 ul total. A blank was generated by adding 20 ul sterile MQ water. 5, 10, and 15 ul aliquots of each sample were used in the determination of the sample concentration. These samples were added to 980 ul coomasie plus reagent (Pierce) and vortexed and allowed to sit at Room temp for a minimum of 10 min. Samples were read at 595 nm. A polynomial regression (not linear regression) was used to fit the standard curve.
Rehydration of the IPG strips with rehydration buffer and samples
For 17cm IPG strips:
Add enough protein sample to get 50 ug and a final volume of 300 ul in rehydration buffer 1
Samples were incubated 30 min at 37C and applied to a 17cm IPG strip face up in the rehydration tray. These trays are disposible.
The tray was wrapped in parafilm and allowed to incubate at room temp overnight.
IEF Focusing gel
17 cm ready strip pH 4-7 Biorad
Strip rehydrated with the control protein sample was cut (one edge) to identify it later.
Paper tabs were placed over the electrodes and a small amount of ddH20 was added to the pre-cut paper tabs.
The bottom of the IPG strip was blotted to remove excess sample (not the gel side).
The IPG strip was placed in the focusing tray gel side down aligning the + with the + electrode.
Approxiamately 2.5ml of mineral oil was overlaid onto the strips and the strips were focused overnight using a rapid focusing program.
Optional: IPG strips can be frozen @ -80 prior to equilibration step. Wrap the tray in parafilm and freeze.
Equilibration step
Biorad Equilibration buffers I and II prepared fresh. Iodoacetamide added to buffer II.
Strips transferred to equilibration tray gel side up, blot the strips to remove mineral oil. 6ml of buffer I added to each well and placed on the orbital shaker for 20 min. 6ml of buffer II added to each well and placed on the orbital shaker for 20 min.
Second Dimension:
Run the gel
Biorad recommends following parameters:
16 mA/ gel through the stacking (30 min)
24 mA/ gel for approx 5 hr
This usually takes longer, so run overnight.
Once the gel is complete, silver stain using Alex’s Protocol.
Scan using the Epson scanner, transparency option.
Adapted from Hai Nguyen and Brad Borlee’s methods.
Wednesday, September 26, 2007
HK